Overview

This notebook analyzes differentially expressed transposable elements (specifically, >6kbp L1HS-L1PA3) and human endogenous retroviruses (HERVs) across brain regions and cell types in the ASAP-PMDBS snRNA-seq dataset.

It builds on the heatmap visualization notebook (TE_expression_heatmaps.Rmd) by using the preprocessed TE pseudobulk data and shared functions to: - Run UMAPs of pseudobulk TE expression. - Identify TE and HERV markers for specific cell types and clusters. - Visualize TE/HERV markers with boxplots across cell types. - Extend analysis to region-specific differential expression.

Data & Functions

# Load preprocessed data and functions
load("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_snRNAseq/visualization/preprocessed_TE_data.RData")
source("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_snRNAseq/visualization/TE_heatmap_functions.R")

Set up

library(umap)
library(DESeq2)
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library(openxlsx)

L1 expression normalization and UMAP

# Load ranked L1s per region (from bulk RNA-seq reference)
L1s_sorted_pfc <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_FL_L1HS_L1PA3_expressed_sorted_mean_expression_PFC.tab")$V1
L1s_sorted_put <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_FL_L1HS_L1PA3_expressed_sorted_mean_expression_PUT.tab")$V1
L1s_sorted_sn <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_FL_L1HS_L1PA3_expressed_sorted_mean_expression_SN.tab")$V1
L1s_sorted_amy <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_FL_L1HS_L1PA3_expressed_sorted_mean_expression_AMY.tab")$V1

# Normalize snRNA-seq pseudobulk counts for these elements
TE_data_norm <- asap_heatmap_snRNA(region = c("PFC", "PUT", "SN", "AMY"), tes = unique(c(L1s_sorted_pfc, L1s_sorted_put, L1s_sorted_sn, L1s_sorted_amy)), filter_low_pseudocounts = F, return_df = T, data = TE_data, title = ">6kbp L1HS-PA3")

# UMAP projection
L1_umap <- umap(t(TE_data_norm$norm_counts[unique(c(L1s_sorted_pfc, L1s_sorted_put, L1s_sorted_sn, L1s_sorted_amy)),TE_data_norm$samplesheet$sample_cluster]))
L1_umap$layout <- as.data.frame(L1_umap$layout)
colnames(L1_umap$layout) <- c("UMAP_1", "UMAP_2")

# Merge metadata and plot UMAP by cell type and diagnosis
L1_umap$layout <- merge(L1_umap$layout, TE_data_norm$samplesheet[,c("sample_cluster", "celltype", "Dx")], by.x="row.names", by.y="sample_cluster")
ggplot(L1_umap$layout, aes(x=UMAP_1, y=UMAP_2, color=celltype)) + geom_point() + theme_bw()

ggplot(L1_umap$layout, aes(x=UMAP_1, y=UMAP_2, color=Dx)) + geom_point() + theme_bw()

HERV expression normalization and UMAP

# Load ranked HERVs per region (from bulk RNA-seq reference)
HERVs_sorted_pfc <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_fl_hervs_expressed_sorted_mean_expression_PFC.tab")$V1
HERVs_sorted_put <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_fl_hervs_expressed_sorted_mean_expression_PUT.tab")$V1
HERVs_sorted_sn <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_fl_hervs_expressed_sorted_mean_expression_SN.tab")$V1
HERVs_sorted_amy <- read.table("/Volumes/MyPassport/ASAP/code/ASAP_PMDBS_bulkRNAseq/results/tables/expressed_fl_hervs_expressed_sorted_mean_expression_AMY.tab")$V1

# Normalize pseudobulk counts for HERVs
HERV_data_norm <- asap_heatmap_snRNA(region = c("PFC", "PUT", "SN", "AMY"), tes = unique(c(HERVs_sorted_pfc, HERVs_sorted_put, HERVs_sorted_sn, HERVs_sorted_amy)), data = HERV_data, return_df = T, title = "HERVs", prefix="HERV", filter_low_pseudocounts = F)

# UMAP projection by cell type and diagnosis
HERV_umap <- umap(t(HERV_data_norm$norm_counts[,-ncol(HERV_data_norm$norm_counts)]))
HERV_umap$layout <- as.data.frame(HERV_umap$layout) 
colnames(HERV_umap$layout) <- c("UMAP_1", "UMAP_2")
HERV_umap$layout <- merge(HERV_umap$layout, HERV_data_norm$samplesheet[,c("sample_cluster", "celltype", "Dx")], by.x="row.names", by.y="sample_cluster")

ggplot(HERV_umap$layout, aes(x=UMAP_1, y=UMAP_2, color=celltype)) + geom_point() + theme_bw()

ggplot(HERV_umap$layout, aes(x=UMAP_1, y=UMAP_2, color=Dx)) + geom_point() + theme_bw()

Differential Expression Analysis: Cell type markers

Function FindTEMarkers()

Wrapper to run DESeq2 comparing each cluster vs all other clusters * Runs DESeq2 for each cluster vs all others. * Stores results in a per-cluster list. * Applied separately to HERVs and L1s.

FindTEMarkers <- function(df, clusters, coldata){
  dds_list <- list()
  res_list <- list()
  for(c in clusters){
    coldata$condition <- ifelse(coldata$cluster == c, paste("Cluster", c), "Other")
    dds_list[[c]] <- DESeqDataSetFromMatrix((df[,coldata$sample_cluster]+1), coldata, design =  ~ condition)
    dds_list[[c]]$condition <- relevel(dds_list[[c]]$condition, "Other")
    dds_list[[c]] <- DESeq(dds_list[[c]])
    res_list[[c]] <- as.data.frame(results(dds_list[[c]]))
    res_list[[c]]$TE_id <- rownames(res_list[[c]])
  }
  return(res_list)
}

HERV_clusters_res_list <- FindTEMarkers(df = HERV_data$te_counts[unique(c(HERVs_sorted_pfc, HERVs_sorted_put, HERVs_sorted_sn, HERVs_sorted_amy)), HERV_data_norm$samplesheet$sample_cluster], clusters = as.character(0:7), coldata =  HERV_data_norm$samplesheet)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
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## estimating size factors
##   Note: levels of factors in the design contain characters other than
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## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
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## final dispersion estimates
## fitting model and testing
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## -- replacing outliers and refitting for 84 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
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## converting counts to integer mode
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## estimating size factors
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## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
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## final dispersion estimates
## fitting model and testing
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## -- replacing outliers and refitting for 105 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
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## converting counts to integer mode
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## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
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## final dispersion estimates
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## -- replacing outliers and refitting for 90 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
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## estimating dispersions
## fitting model and testing
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## converting counts to integer mode
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## estimating dispersions
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## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
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##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
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## final dispersion estimates
## fitting model and testing
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## -- replacing outliers and refitting for 117 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
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## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
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##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 87 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
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## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 72 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 96 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 93 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
L1_clusters_res_list <- FindTEMarkers(df = TE_data$te_counts[unique(c(L1s_sorted_pfc, L1s_sorted_put, L1s_sorted_sn, L1s_sorted_amy)), TE_data_norm$samplesheet$sample_cluster], clusters = as.character(0:7), coldata =  TE_data_norm$samplesheet)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 23 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 27 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 32 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 31 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 32 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 27 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
write.xlsx(HERV_clusters_res_list, "/Volumes/MyPassport/ASAP/data/ASAP_PMDBS_snRNAseq/results/tables/HERV_markers_clusters_res.xlsx")
write.xlsx(L1_clusters_res_list, "/Volumes/MyPassport/ASAP/data/ASAP_PMDBS_snRNAseq/results/tables/L1_markers_clusters_res.xlsx")

Cell type Markers visualization

plot_topn_markers <- function(df, res, cluster, prefix, samplesheet, ytitle, ylim = NA, title = NA, topn = 1, colors = NA){
    if(is.numeric(topn)){
      signif_res <- res[[cluster]][which(res[[cluster]]$log2FoldChange > 1 & res[[cluster]]$padj < 0.05),]
      marker_names <- rownames(signif_res[order(signif_res$log2FoldChange, signif_res$baseMean),])[topn]  
    }else{
      marker_names <- topn
    }
    
    
    df <- as.data.frame(t(df[marker_names,]))
    for(te in colnames(df)[startsWith(colnames(df), prefix)]){
      df[,te] <- as.numeric(df[,te])  
    }
    df <- merge(df, samplesheet, by.x="row.names", by.y="sample_cluster")
    df_melt <- reshape2::melt(df[,c("Row.names", marker_names, "celltype", "cluster")], by=list(df$Row.names, df$celltype))
    
    plt <- ggplot(df_melt, aes(x=celltype, y=value, fill=celltype)) + 
      geom_jitter(width = 0.2, alpha=0.5, aes(color = celltype)) +
      geom_boxplot(outliers = F, alpha=0.5) +
      theme_bw() + labs(y = ytitle, x="") + # Normalized expression? Log2? Specify
      theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) + ggtitle(marker_names)
    
    pval <- format(res[[cluster]][marker_names, "pvalue"], digits = 2, scientific = T)
    celltype_pval <- unique(df_melt[which(df_melt$cluster == cluster), "celltype"])
    if(!all(is.na(ylim))){
      plt <- plt + coord_cartesian(ylim = c(0 , ylim))
      plt <- plt + annotate("text", label = paste("p =", pval), x=celltype_pval, y=ylim-(ylim*0.1), fill=NA)
    }else{
      plt <- plt + annotate("text", label = paste("p =", pval), x=celltype_pval, y=max(plt$data$value), fill=NA)
    }
    
    if(!all(is.na(title))){
      plt <- plt + ggtitle(paste(title, marker_names, sep = ": "))
    }
    
    if(!all(is.na(colors))){
      plt <- plt + scale_color_manual(values = colors) + scale_fill_manual(values = colors)
    }
    
    return(plt)
}

celltype_colors <- c("0_ExcNeurons" = "#8dd1c5",
                     "1_InhNeurons" = "#fae447",
                     "2_Astrocytes" = "#bdb9d9",
                     "3_OPC" = "#f47f73",
                     "4_Microglia" = "#81b1d3",
                     "5_Oligodendrocytes" = "#fbb464",
                     "6_VLMC" = "#b4d66c",
                     "7_Tcells" = "#f9cde1")

HERV cell-markers examples

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "0", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup215", ylim = 1.5, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Exc neurons")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "0", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup1520", ylim = 1.5, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Exc neurons")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "1", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup2680",ylim = 0.1,  ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Inh neurons")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "1", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup1556",ylim = NA,  ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Inh neurons")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "2", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup1001",ylim = 2, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Astrocytes")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "5", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup2376", ylim = 5,ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Oligodendrocytes")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "3", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup2739", ylim = 1, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "OPCs")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "4", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup1359", ylim = 7.5,  ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Microglia")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "6", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup1776", ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "VLMCs")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_res_list, cluster = "7", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup876",ylim = 3, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "T cells")
## Warning in plot_topn_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

L1s cell-markers examples

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "0", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA3_dup10072", ylim = 2, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Exc neurons")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "1", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA3_dup3102", ylim = 2, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Inh neurons")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "3", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA2_dup2091", ylim = NA, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "OPCs")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "4", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA3_dup7550",  ylim = 1, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Microglia")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "5", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA2_dup1778", ylim = 2.5, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Oligodendrocytes")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

plot_topn_markers(df = TE_data_norm$norm_counts, res = L1_clusters_res_list, cluster = "2", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = "L1PA2_dup1933",ylim = 4, ytitle = "log2(Normalized Expression)", colors = celltype_colors, title = "Astrocytes")
## Warning in plot_topn_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_res_list, : NAs introduced by coercion
## Using Row.names, celltype, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = celltype_pval, :
## Ignoring unknown parameters: `fill`

Region-specific DE analysis

Function: FindTEMarkers_region()

  • Extends DESeq2 test to cluster-region combinations
  • Identifies markers enriched in specific brain regions (e.g. microglia in SN vs AMY)
FindTEMarkers_region <- function(df, clusters,regions, coldata){
  dds_list <- list()
  res_list <- list()
  for(c in clusters){
    dds_list[[c]] <- list()
    res_list[[c]] <- list()
    for(r in regions){
      coldata$condition <- ifelse(coldata$cluster == c & coldata$Region == r, paste("Cluster", c, ", region", r), "Other")
      dds_list[[c]][[r]] <- DESeqDataSetFromMatrix((df[,coldata$sample_cluster]+1), coldata, design =  ~ condition)
      dds_list[[c]][[r]]$condition <- relevel(dds_list[[c]][[r]]$condition, "Other")
      dds_list[[c]][[r]] <- DESeq(dds_list[[c]][[r]])
      res_list[[c]][[r]] <- results(dds_list[[c]][[r]])
      res_list[[c]][[r]] <- as.data.frame(results(dds_list[[c]][[r]]))
      res_list[[c]][[r]]$TE_id <- rownames(res_list[[c]][[r]])
    }
  }
  return(res_list)
}

HERV_clusters_region_res_list <- FindTEMarkers_region(df = HERV_data$te_counts[unique(c(HERVs_sorted_pfc, HERVs_sorted_put, HERVs_sorted_sn, HERVs_sorted_amy)), HERV_data_norm$samplesheet$sample_cluster], clusters = as.character(0:7), regions = c("SN", "AMY", "PFC", "PUT"), coldata =  HERV_data_norm$samplesheet)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 89 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 90 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 70 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 86 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 112 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 96 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 83 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 115 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 93 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 90 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 78 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 91 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 112 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 98 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 90 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 135 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 100 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 89 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 85 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 89 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 77 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 83 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 82 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 84 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 103 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 98 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 92 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 92 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 95 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 91 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 88 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 92 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
L1_clusters_region_res_list <- FindTEMarkers_region(df = TE_data$te_counts[unique(c(L1s_sorted_pfc, L1s_sorted_put, L1s_sorted_sn, L1s_sorted_amy)), TE_data_norm$samplesheet$sample_cluster], clusters = as.character(0:7), regions = c("SN", "AMY", "PFC", "PUT"), coldata =  TE_data_norm$samplesheet)
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 27 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 22 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 20 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 34 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 25 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 24 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 24 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 27 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 28 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 23 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 32 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 37 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 25 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 33 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 30 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 25 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 39 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 30 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 28 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 33 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 28 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 28 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 26 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating size factors
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## estimating dispersions
## gene-wise dispersion estimates
## mean-dispersion relationship
## -- note: fitType='parametric', but the dispersion trend was not well captured by the
##    function: y = a/x + b, and a local regression fit was automatically substituted.
##    specify fitType='local' or 'mean' to avoid this message next time.
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## final dispersion estimates
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
## -- replacing outliers and refitting for 29 genes
## -- DESeq argument 'minReplicatesForReplace' = 7 
## -- original counts are preserved in counts(dds)
## estimating dispersions
## fitting model and testing
##   Note: levels of factors in the design contain characters other than
##   letters, numbers, '_' and '.'. It is recommended (but not required) to use
##   only letters, numbers, and delimiters '_' or '.', as these are safe characters
##   for column names in R. [This is a message, not a warning or an error]
write.xlsx(HERV_clusters_region_res_list, "/Volumes/MyPassport/ASAP/data/ASAP_PMDBS_snRNAseq/results/tables/HERV_markers_clusters_region_res.xlsx")
write.xlsx(L1_clusters_region_res_list, "/Volumes/MyPassport/ASAP/data/ASAP_PMDBS_snRNAseq/results/tables/L1_markers_clusters_region_res.xlsx")

Function: plot_region_markers()

  • Plots region-specific markers for each cell type.
  • Highlights elements uniquely enriched in one region but not the others.
plot_region_markers <- function(df, res, cluster, region, prefix, samplesheet, ytitle, ylim = NA, title = NA, topn = 1, colors = NA){
    if(is.numeric(topn)){
      signif_res <- res[[cluster]][[region]][which(res[[cluster]][[region]]$log2FoldChange > 1 & res[[cluster]][[region]]$padj < 0.05),]
      marker_names <- rownames(signif_res[order(signif_res$log2FoldChange, signif_res$baseMean),])
      signif_regions <- data.frame(marker = marker_names)
      for(r in names(res[[cluster]][names(res[[cluster]]) != region])){
        r_res <- res[[cluster]][[r]]
        signif_regions[,r] <- r_res[marker_names, "log2FoldChange"] > 0 & r_res[marker_names, "padj"] < 0.05
      }
      signif_regions <- signif_regions[which(!apply(signif_regions[,-1], 1, any)),]
      signif_res <- signif_res[signif_regions$marker,]
      marker_names <- rownames(signif_res[order(signif_res$log2FoldChange, signif_res$baseMean)[topn],])
    }else{
      marker_names <- topn
    }
    
    
    df <- as.data.frame(t(df[marker_names,]))
    for(te in colnames(df)[startsWith(colnames(df), prefix)]){
      df[,te] <- as.numeric(df[,te])  
    }
    df <- merge(df, samplesheet, by.x="row.names", by.y="sample_cluster")
    df_melt <- reshape2::melt(df[,c("Row.names", marker_names, "celltype", "Region", "cluster")], by=list(df$Row.names, df$celltype))
    celltype <- unique(df_melt$celltype)
    df_melt <- df_melt[which(df_melt$cluster == cluster),]
    plt <- ggplot(df_melt, aes(x=Region, y=value, fill=Region)) + 
      geom_jitter(width = 0.2, alpha=0.5, aes(color = Region)) +
      geom_boxplot(outliers = F, alpha=0.5) +
      theme_bw() + labs(y = ytitle, x="") + # Normalized expression? Log2? Specify
      ggtitle(paste(celltype, marker_names, sep=": "))
    pval <- format(res[[cluster]][[region]][marker_names, "pvalue"], digits = 2, scientific = T)
    if(!all(is.na(ylim))){
      plt <- plt + coord_cartesian(ylim = c(0 , ylim))
      plt <- plt + annotate("text", label = paste("p =", pval), x=region, y=ylim-(ylim*0.1), fill=NA)
    }else{
      plt <- plt + annotate("text", label = paste("p =", pval), x=region, y=max(plt$data$value), fill=NA)
    }
    
    if(!all(is.na(title))){
      plt <- plt + ggtitle(paste(title, marker_names, sep = ": "))
    }
    
    if(!all(is.na(colors))){
      plt <- plt + scale_color_manual(values = colors) + scale_fill_manual(values = colors)
    }
    
    return(plt)
}

Some region-specific examples

plot_region_markers(df = TE_data_norm$norm_counts, res = L1_clusters_region_res_list, cluster = "5",region = "SN", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = 4, ytitle = "log2(Normalized Expression)", title = "Oligodendrocytes in SN", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y =
## max(plt$data$value), : Ignoring unknown parameters: `fill`

plot_region_markers(df = TE_data_norm$norm_counts, res = L1_clusters_region_res_list, cluster = "0",region = "AMY", prefix = "L1", samplesheet = TE_data_norm$samplesheet, topn = 2, ytitle = "log2(Normalized Expression)", title = "EN in Amygdala", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = TE_data_norm$norm_counts, res =
## L1_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y =
## max(plt$data$value), : Ignoring unknown parameters: `fill`

plot_region_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_region_res_list, cluster = "0", region = "PUT", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = 1,ytitle = "log2(Normalized Expression)", title = "EN in Putamen", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y =
## max(plt$data$value), : Ignoring unknown parameters: `fill`

plot_region_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_region_res_list, cluster = "0", region = "PUT", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = "HERV_dup398",ytitle = "log2(Normalized Expression)", title = "EN in Putamen", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y =
## max(plt$data$value), : Ignoring unknown parameters: `fill`

plot_region_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_region_res_list, cluster = "3", region = "SN", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet, topn = 1,ytitle = "log2(Normalized Expression)", title = "OPCs in SN", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y =
## max(plt$data$value), : Ignoring unknown parameters: `fill`

plot_region_markers(df = HERV_data_norm$norm_counts, res = HERV_clusters_region_res_list, cluster = "3", region = "AMY", prefix = "HERV", samplesheet = HERV_data_norm$samplesheet,ylim = 0.5, topn = 1,ytitle = "log2(Normalized Expression)", title = "OPCs in AMY", colors = rep("lightgrey", 4))
## Warning in plot_region_markers(df = HERV_data_norm$norm_counts, res =
## HERV_clusters_region_res_list, : NAs introduced by coercion
## Using Row.names, celltype, Region, cluster as id variables
## Warning in annotate("text", label = paste("p =", pval), x = region, y = ylim -
## : Ignoring unknown parameters: `fill`

Outputs

Notes

  • This notebook should be run after TE_expression_heatmaps.Rmd, since it uses preprocessed pseudobulk data and helper functions.